Autophagy is the principle recycling center of the cell, where bulk cytoplasmic material and damaged organelles are engulfed by double membrane vesicles coined autophagosomes that then fuse with lysosomes, which degrade their cargo and recoup building blocks and energy, both as a homeostatic mechanism and as a survival strategy during times of stress. Defects in the autophagy pathway can lead to several pathologies including degenerative diseases and cancer. Importantly, using genetic approaches, we have shown that inhibiting the autophagy pathway disables several types of refractory human malignancies and augments the efficacy of irradiation and cancer therapeutics. Selective modulators of the autophagy pathway are clearly needed yet none exist. The autophagy pathway is controlled by a conserved serine/threonine kinase coined Ulk1 (UNC-51-like kinase-1). Our Multi-PI research team has shown that: (1) Ulk1 kinase activity is essential for activating the autophagy pathway; (2) Atg13 is a bona fide substrate of Ulk1; and (3) that Ulk1-directed phosphorylation of Atg13 on S318 is essential for autophagy. Given the importance of Ulk1 as a target we developed a series of high-throughput compatible assays and are now positioned to perform a HTS-campaign, where we will implement a rigorous research operating plan comprised of biochemical and cell-based assays to identify, confirm and validate selective small molecule Ulk1 inhibitors. Specifically, we have used our novel research tools to develop a 384-well compatible, homogenous Ulk1 biochemical assay. Further, we have validated this assay using the Sigma-LOPAC (Library of Pharmacologically Active Compounds) library. In the studies of Aim 1, we will miniaturize this assay to a 1536-well format and perform a HTS campaign against the 650,000 small molecule compound library of The Scripps Research Institute. In Aim 2 'hits' identified in the primary screen will be confirmed and further evaluated to: (i) triage false positives; (ii) confirm 'hits' using an orthogonal biochemical assay (iii) rank order the biochemical activity of 'hits' based upon the concentration needed to inhibit 50% (IC50) of Ulk1 kinase activity; and (iv) triage scaffolds for chemical tractability prior to cel-based assay analyses. In Aim 3, lead compounds will be assessed using a series of validated follow-up assays. First, cellular potency of confirmed HTS 'hits' will be determined using a quantitative, cell-based, Atg13S318 phosphorylation assay. Second, we will define the mechanism of inhibitor action of leads (ATP competitive, substrate competitive, etc.). Third, we will determine the selectivity of top leads against a panel of 456 kinases (DiscoveRx) including the highly related kinase Ulk2. Finally, our most active leads will be tested in cell-based functional assays, where we will: (i) confirm that lead molecules inhibit autophagy using validated luciferase-LC3, luciferase-p62/SQSTM1 and mCherry-GFP-LC3 autophagic flux assays; and (ii) assess the biological effects of lead inhibitors in primary wild type and Ulk1- an Atg7-deficient mouse embryo fibroblast and myeloid cells treated with agents that activate the autophagy pathway.